The construction of chimeric plasmids containing the complete genome of mouse L cell mitochondria has made it possible to purify large quantities of mtDNA sequences and develop a fine structure physical map of the genome using restriction endonucleases. The plasmid DNA also provides sufficient template for in vitro transcription and translation mtDNA sequences using eukaryote RNA polymerases and protein synthesizing extracts. Mutant L cells resistant to chloramphenicol, oligomycin and ethidium bromide have been isolated. Sequence changes in mtDNA isolated from these mutants can be detected using restriction endonucleases and heteroduplex analysis by S1 nuclease digestion or electron microscopy. By mapping the sites of these mutations and correlating them with changes in mitochondrial enzyme or ribosome activities we will further our understanding of the geneetic function of mammalian mitochondrial DNA.